Induced pluripotent stem cells (iPSCs) hold enormous promise for cell therapy in tissue engineering and for the study of disease. This promise is due to the ability to create pluripotent, embryonic stem-like cells from any genetic source. Patient-specific immuno-matched cells can be chosen as the parent cell line for reprogramming into iPSCs. The iPSCs are then used to create cells and tissues that a patient tolerates without rejection. The iPSCs can also be derived from patients with genetic diseases for use in the discovery of disease mechanisms and treatments. Like other pluripotent stem cells, iPSCs can be used to create heart, brain, retina, liver and cells of other tissues for testing drug candidates, assessing efficacy of drugs, and/or uncovering toxic effects of drug candidates at an earlier state in the drug development process.
It is possible to derive iPSCs from differentiated cells, for example, by using transcription factors to turn on genes that were expressed in stem cells but later silenced in the differentiated cells. Gene silencing represents one type of epigenetic change, or change to phenotype or gene expression caused by mechanisms beyond the underlying DNA sequence. If epigenetic changes associated with cell differentiation could be reversed, then the frequency and rate of pluripotent cell induction could be increased, and the safety and efficiency of producing iPSC colonies would be improved. For example, safety can be improved by reducing the probability of producing cancerous cells (Okita et al., 2007).
In addition, there remains a need for determining whether and when iPSCs have been successfully derived from differentiated cells. Because there are distinct intermediate stages between the differentiated and pluripotent states which differ in morphology, it is possible to image the cell population and use mathematical image analysis to determine which state the cells in the image have reached. In combination with epigenetic manipulations of cells, the image analysis offers a powerful method for deriving and evaluating a useful population of iPSCs with increased speed and efficacy.
A scalable, verifiable method for identifying pluripotency in a non-destructive and non-invasive manner would be ideal for deriving rare iPSCs from somatic cells or for quality control of large numbers of hESC colonies intended for cell therapy. At present, methods for human hESC classification are limited to visual inspection of live cells by a trained microscopist or biochemical or immunochemical staining. While visual observation using brightfield or phase contrast microscopy is non-invasive, it is time consuming, non-quantitative, and cannot be scaled up for the large quantity of cells expected in a therapeutic or commercial setting. Likewise, while biochemical staining of hESCs is consistent, quantitative, and automatable, it is destructive and renders the sample unfit for therapeutic use (Sammak et al., 2008). Live cell fluorescent markers can be used to recognize nascent iPSC colonies (Chan et al., 2009) but are invasive, requiring addition of extracellular or membrane permanent dyes that have limited application in kinetic assays because of dye loss over time. Furthermore, fluorescent dyes and methods for detecting such dyes may damage photosensitive cells. Further, accurate quality control requires a measure of the homogeneity of cell morphology, which is nearly impossible to perform visually for a very large numbers of cell culture plates. In contrast, morphological measurements could serve as end-point indicators of cell pluripotency or differentiation and provide real-time measurement of the experimental agents on cells.